Dry culture media

ABSTRACT

A device for culturing microorganisms is described comprising a body member comprising a self-supporting, water-proof substrate; a layer of adhesive coated on the substrate, the adhesive being non-inhibitory to the growth of microorganisms; and a cold-water-soluble powder adhered uniformly to the surface of the adhesive, the powder comprising a gelling agent and/or nutrients for growing microorganisms. Another device is described comprising a body member comprising a self-supporting, water-proof substrate; a coating coated directly on the substrate, the coating being substantially water-free and consisting essentially of a cold-water-reconstitutable material comprising a gelling agent and/or nutrients for growing microorganisms; and a cover sheet releasably adhered to the bottom member, the cover sheet being substantially impermeable to bacteria and water vapor. The devices are activated by the addition of water or an aqueous test sample.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. applicationSer. No. 228,893, which was filed on Jan. 27, 1981 and is now abandoned.

FIELD OF THE INVENTION

This invention relates to a device for culturing microorganisms. Inparticular, it relates to a device containing a culture medium in a formwhich is cold-water-reconstitutable. When contacted with water, themedium forms a substantially homogeneous medium without mixing.

BACKGROUND ART

Media for culturing bacteria are generally prepared by dispersing asolidifying agent in an aqueous solution containing nutrients and otheringredients necessary for the growth of specific microorganisms.Unfortunately, use of conventional solidifying agents is ofteninconvenient for the end-user. For example, when carrying out standard"plate count" or "pour plate" methods to determine the number ofmicroorganisms in a liquid sample such as water or milk, the use ofconventional agar medium is particularly inconvenient andtime-consuming. The agar medium, which has generally been prepared inbulk and sterilized ahead of time, must be melted in boiling water or byexposure to flowing steam. The hot agar must then be carefully cooled toapproximately 45° C. prior to pouring into petri dishes. A series ofdilutions of the test sample is then prepared and an aliquot of eachdilution is placed in a petri dish. The cooled, but still liquified,agar medium is then poured into each dish, mixed with the aliquot oftest sample, swirled to mix and allowed to solidify. After incubation,the number of colonies growing in each dish are counted by visualinspection. In this manner the number of microorganisms orcolony-forming units present in the test sample can be determined.

It is apparent from the foregoing description that a simpler method ofobtaining standard plate counts is desirable, particularly one thateliminates the need for the end-user to melt and cool the agar mediumand pour it into the petri dishes.

The prior art has provided several gelling agents for microbiologicalgrowth media which are rehydratable at room temperature. For example,U.S. Pat. No. 3,046,201 suggests the use of certain polyacrylamides asgelling agents. U.S. Pat. No. 3,360,440 describes a microbiologicalmedium in which the gelling agent is a cold-water-soluble modifiedcellulose. The aforementioned gelling agents are prepared by specialprocesses involving expensive lyophilization procedures to increase thesurface area of the dry powder to render it more easily rehydrated. Whenrehydrated, mixing is generally required to obtain a homogeneous gel.

U.S. Pat. No. 3,881,993 describes a device for assaying liquid specimensfor microorganisms. One embodiment of the device comprises filter paperwhich is impregnated with a gelling agent and nutrients for growingmicroorganisms and which is adhered to a film by means of an adhesivelayer. This embodiment suffers from the disadvantage that it isgenerally only semi-quantitative, due possibly to the presence of thefilter paper. It is believed the filter paper is not suitablytransparent and that it therefore renders counting of bacterial coloniesdifficult. Also, presence of the filter paper renders isolation ofindividual bacterial colonies impractical.

SUMMARY OF THE INVENTION

The present invention provides a preferred device for growingmicroorganisms, comprising: a bottom member comprising a self-supportingwater-proof substrate having upper and lower surfaces; a layer ofadhesive coated on the upper surface of the substrate, the adhesivebeing noninhibitory to the growth of microorganisms; and a coating ofcold-water-soluble powder adhered uniformly to the surface of theadhesive, the powder comprising at least one ingredient selected fromthe group consisting of a gelling agent, one or more nutrients forgrowing microorganisms and a mixture thereof. Preferably, the devicefurther comprises a cover sheet releasably adhered to at least a portionof the body member to prevent contamination of the device during storageand incubation.

The present invention also provides a device for growing microorganisms,comprising: a bottom member comprising a self-supporting, water-proofsubstrate having upper and lower surfaces with a coating adhered to atleast a portion of the upper surface, the coating being substantiallywater-free and consisting essentially of a cold-water-reconstitutablematerial comprising at least one ingredient selected from the groupconsisting of a gelling agent, one or more nutrients for growingmicroorganisms, and a mixture thereof; and a cover sheet releasablyadhered to at least a portion of the bottom member, the cover sheetbeing substantially impermeable to bacteria and water vapor.

If a gelling agent is present in the coating ofcold-water-reconstitutable material (the cold-water-soluble powder inthe preferred device), it is preferably present in an amount sufficientto form a substantially transparent gel having a Brookfield viscosity ofat least 1500 cps. In the preferred embodiment, a dye is also includedin the coating of cold-water-reconstitutable material. The dye issoluble in the aqueous medium so that it can react with the growingmicroorganisms and enables better visualization of the bacterialcolonies.

The means for covering the substrate to prevent contamination duringincubation is preferably a sheet attached in hinge-like fashion to oneend of the body member. The cover sheet is simply peeled back, and theliquid sample placed on the substrate. The cover sheet is then returnedto its original position thereby sealing in the gelled medium. The coversheet is preferably transparent to allow the bacterial colonies to beseen. Optionally, the surface of the cover sheet contacting thesubstrate may have a coating of cold-water-reconstitutable materialadhered thereto, that coating containing a gelling agent and/ornutrients for growing microorganisms. The materials used to form thecover sheet may be conveniently selected to obtain the desiredpermeability to gases such as oxygen.

When a predetermined amount of water or other aqueous test sample isplaced on the substrate in contact with the coating ofcold-water-reconstitutable material, the gelling agent preferablycontained in that coating immediately hydrates in the sample along withthe other dry ingredients adhered to the substrate and forms a gelledmedium. No mixing is required. There is no need for the end-user to heatthe medium or otherwise treat it to obtain a homogeneous gel.

The devices of the invention provide a marked improvement over prior artdevices and techniques for carrying out standard pour plate methods aswell as other microbiological testing. The coatings of medium of thedevices of the present invention do not contain matrixes which wouldadversely affect one's ability to visualize and isolate bacterialcolonies. Not only will the medium provided by the device allowenumeration of the bacterial colonies growing in the medium, but thecolonies may be easily isolated for further testing in the same manneras bacterial colonies growing on conventional agar medium in a petridish. The devices have the added feature of being much more compact andlight-weight than petri dishes and take up less space in the laboratory.Furthermore, the devices are completely disposable allowing for saferand more rapid clean-up after use. The preferred devices of the presentinvention (i.e., those comprising a cold-water-soluble powder whichcomprises a gelling agent) provide results comparable to those providedby conventional pour plates.

DESCRIPTION OF THE DRAWINGS

The invention may be further illustrated by reference to theaccompanying drawings wherein:

FIG. 1 is a top perspective view, partially in section, of a preferredmicrobiological growing device of the invention;

FIG. 2 is a top perspective view of an alternative embodiment of theinvention;

FIG. 3 is a cross sectional view of device of FIG. 1;

FIG. 4 is a top view of the device of FIG. 2 showing a grid patternprinted on the substrate.

FIGS. 1 and 4 illustrate a preferred device in accordance with thepresent invention. The microbiological growing device 10 includes a bodymember comprising a self-supporting water-proof substrate 12 havingupper and lower surfaces. Substrate 12 is preferably a relatively stifffilm of a material such as polyester, polypropylene or polystyrene whichwill not absorb or otherwise be affected by water. Polyester filmsapproximately 0.004 to 0.007 inch thick, polyproplyene filmsapproximately 0.004 to 0.008 inch thick and polystyrene filmsapproximately 0.015 inch thick have been found to work well. Othersuitable substrates include paper with a polyethylene or otherwater-proof coating. An example of a suitable polyethylene-coated papersubstrate is "Schoeller Type MIL" photoprint paper (commerciallyavailable from Schoeller Pulaski, New York). The substrate 12 may beeither transparent or opaque, depending on whether one wishes to viewbacterial colonies through the substrate. To facilitate the counting ofbacterial colonies, the substrate 12 preferably has a square gridpattern printed thereon as shown in FIG. 4.

Substrate 12 is coated on its upper surface with a layer of an adhesive14 which serves to hold the dry gelling agent and/or nutrients in auniform monolayer for easy hydration. Adhesive 14 must bewater-insoluble and non-inhibitory to the growth of microorganisms.Preferably, the adhesive is sufficiently transparent when wet to enablethe viewing of bacterial colonies through the film coated with theadhesive. It is preferred that adhesive 14 be pressure-sensitive.However, heat-activated adhesives wherein a lower melting substance iscoated onto a higher melting substance may also be used. Water-activatedadhesives such as mucilage may also be useful.

Adhesive 14 should be coated onto substrate 12 in a thickness which ispreferably less than the diameter of the particles of the powderedgelling agent and/or nutrients. The object is to apply enough adhesiveto adhere the particles to the substrate but not so much that theparticles become completely embedded in the adhesive. A uniformmonolayer of powder 16 is desired with sufficient surface area exposedfor hydration. Generally, an adhesive layer in the thickness range of0.0002 to 0.0005 inch is suitable.

The presently preferred adhesive is a copolymer ofisooctylacrylate/acrylamide (in a mole ratio of 94/6). Other pressuresensitive adhesives which may be used include isooctylacrylate/acrylicacid (in a mole ratio of 95/5 or 94/6) and silicone rubber. Adhesiveswhich turn milky upon exposure to water are less preferred, but may beused in conjunction with a non-transparent substrate or where colonyvisualization is not required.

A monolayer of cold-water-soluble powder 16 is adhered uniformly toadhesive layer 14. Powder 16 comprises at least one ingredient selectedfrom the group consisting of a gelling agent, one or more nutrients forgrowing microorganisms, and a mixture of a gelling agent and one or morenutrients for growing microorganisms. As used in the specification andclaims, the term "powder" designates a finely divided particulatematerial having an average diameter of less than 400 micrometers. Asused in the specification and claims, the term "cold-water-soluble"designates material which forms a solution in water at room temperature.

The "cold-water-solubility" of the powders employed in the devices ofthe present invention may result, for example, from the inclusion inthese powders of an appropriate gelling agent. Suitable gelling agentsfor inclusion in powder 16 include both natural and synthetic gellingagents which form solutions in water at room temperature. Gelling agentssuch as hydroxyethyl cellulose, carboxymethyl cellulose, polyacrylamide,locust bean gum and algin form solutions in water at room temperatureand are suitable gelling agents for providing powders which are"cold-water-soluble." The preferred gelling agents for powder 16 areguar gum and xanthan gum, these gelling agents being useful individuallyor in combination with one another. Nutrients for growing microorganismsform solutions in water at room temperature.

As indicated, powder 16 may comprise only a gelling agent. Where thedevice, as manufactured, contains a powder comprising only gellingagent, the end user adds his own special nutrients "tailored" to thetype of microorganisms he wishes to grow. For example, dry powderednutrients may be suspended in a rapidly-evaporating liquid such asethanol or "Freon". In other instances, dry powdered nutrients may besuspended or dissolved in aqueous solutions. An aliquot of the liquid isadded to the surface of substrate 12 which has been coated previouslywith adhesive and gelling agent. The liquid is allowed to evaporate,leaving ample nutrients along with the gelling agent.

In another embodiment of the invention, powder 16 may comprise nutrientsbut no gelling agent. Gelling agent is only required if one desires tovisualize and/or isolate discrete bacteria colonies. In manymicrobiological tests, such as tests for bacteria identification orantibiotic susceptibility, broth media are used, and there is no needfor a viscous gel. In devices for carrying out such tests, the gellingagent may be omitted.

Where gelling agent is included in powder 16, a sufficient amount of thegelling agent is adhered to the substrate so that a predeterminedquantity of water or an aqueous sample, e.g., 1-3 milliliters, placed onthe substrate will form a gel having a viscosity of about 1500 cps ormore when measured at 60 rpm with a Brookfield Model LVF viscometer at25° C. Gels of this viscosity will allow convenient handling andstacking and provide distinct colony identification. In most cases 0.025to 0.050 gram of guar gum on a surface area of 3.14 sq. inches willprovide a sufficiently viscous gel when hydrated with 1-3 milliliters ofan aqueous sample. The size of the powder particles can be used tocontrol the coating weight per unit area. For example, approximately 100mesh guar gum coats to a weight of about 0.05 grams/2 inch diameterdisc; and a 400 mesh guar gum coats to a weight of about 0.025 grams/2inch diameter disc. If additional amounts of gelling agent and/ornutrients are required, the optional cover sheet of this embodiment mayalso be coated.

The preferred coating mixture for powder 16 is as follows:

15 grams guar gum or xanthan gum

5 grams peptone

2.5 grams yeast extract

1 gram dextrose

0.06 gram sodium carbonate

0.12 gram "Cab-O-Sil M-5" (a fumed silicon dioxide, commerciallyavailable from Cabot Corporation)

Sodium carbonate is employed to provide a medium exhibiting a neutralpH. "Cab-O-Sil M-5" is employed as a processing aid. Of course, theparticular coating mixture used for powder 16 may depend upon the typeof microorganisms to be grown.

In preparing a coating mixture comprising the above ingredients, thepeptone, yeast extract, dextrose and sodium carbonate are dissolved inwater and the resulting solution is spray-dried by conventional means togive a homogeneous mixture of the ingredients. The remaining ingredientsare then combined with the above mixture to provide the final coatingmixture.

It may be desirable to incorporate a dye into the medium mixture.Alternatively, the dye may be incorporated in adhesive 14. Suitable dyesare those which are metabolized by the growing microorganisms, and whichcause the colonies to be colored for easier visualization. Examples ofsuch dyes include triphenyl tetrazolium chloride, p-tolyl tetrazoliumred, tetrazolium violet, veratryl tetrazolium blue and related dyes.Other suitable dyes are those sensitive to pH changes such as neutralred.

For some uses it may be desirable to form a medium stiff enough to allowinoculation of microorganisms by streaking. To form streakable medium,it may be desirable to include a small amount of cross-linking agentpowder 16 where powder 16 includes a gelling agent. For example, withguar gum, cross-linking agents such as potassium tetraborate, aluminumor calcium salts may be added in an amount less than 1.0 percent byweight of powder 16. One must be careful to select a cross-linking agentwhich does not substantially affect the growth of the intendedmicroorganism.

It is also contemplated within the scope of the invention that powder 16may optionally include reagents necessary for carrying out certainmicrobiological tests. For example, antibiotics may be included forcarrying out antibiotic susceptibility tests. For microorganismidentification, reagents such as those which undergo a color change inthe presence of a particular type of microorganism may be included.

In the device of FIG. 1, the body member includes a spacer elementapplied to the upper surface of substrate 12, the spacer elementcomprising a piece of spacer 18 having a circular hole 20 cut throughthe center to expose the particles 16 on substrate 12. The walls of hole20 provide a well of predetermined size and shape to confine the mediumfollowing hydration. Spacer 18 should be thick enough to form a well ofthe desired volume, e.g., 1, 2 or 3 milliliter. Closed cell polyethylenefoam is preferred material for spacer 18, but any material which ishydrophobic (non-wetting), inert to microorganisms, and capable ofwithstanding sterilization may be used.

Adhered to one edge of spacer 18 of the body member is a cover sheet 22.Cover sheet 22 is preferably transparent to facilitate counting of thebacterial colonies and is substantially impermeable to bacteria andwater vapor. As used in the specification and claims, "substantiallyimpermeable to bacteria and moisture vapor" designates cover sheetswhich prevent undesired contamination of the dehydrated medium duringshipping, storage and use of the devices and which provide anenvironment which will support the growth of microorganisms during theincubation period. Generally, it will have the same properties assubstrate 12, but need not be as stiff. Cover sheet 22 can be selectedto provide the amount of oxygen transmission necessary for the type ofmicroorganism desired to be grown. For example, polyester films have alow oxygen permeability (less than 5 g/100 in² /24 hours per 0.001 inchof thickness) and would be suitable for growing anaerobic bacteria. Onthe other hand, polyethylene has a very high oxygen permeability(approximately 500 g/100 in² /24 hours per 0.001 inch of thickness) andwould be suitable for aerobic organisms. The presently preferredmaterial for cover sheet 22 is a 1.6 mil biaxially-orientedpolypropylene film. Cover sheet 22, as illustrated, is coated withoptional layers of adhesive 14' and powder 16'. It is to be understoodthat cover sheet 22 may alternatively be adhered to substrate 12 of thebody member and that it may be free of any coating or may be coated witha layer of pressure-sensitive adhesive only.

The embodiment of FIG. 2 is identical to that of FIG. 1 except thatspacer 18 is not present. A template, such as a weighted circular ring,may be applied temporarily to the outside of cover sheet 22, afterclosing, to confine the gel to a specific region.

Although both of the embodiments illustrated in the drawing have a coversheet 22 attached to the device, it is also contemplated within thescope of the invention that the powder-containing embodiments may beuncovered and simply placed in a sterile environment during storage andincubation.

Another device (not illustrated) in accordance with the presentinvention comprises a bottom member comprising a self-supporting,water-proof substrate having upper and lower surfaces. Coated on atleast a portion of the upper surfaces of the substrate is a coatingwhich is substantially water-free and which consists essentially of acold-water-reconstitutable material comprising at least one ingredientselected from the group consisting of a gelling agent, one or morenutrients for growing microorganisms, and a mixture of a gelling agentand one or more nutrients for growing microorganisms. As used in thespecification and claims, the phrase "substantially water-free"designates a coating which has a water content no greater than about thewater content of the dehydrated coating once it has been permitted toequilibrate with the ambient environment.

Suitable substrates for employment as the body member in this embodimentinclude those discussed above in connection with the illustratedembodiments.

This embodiment also comprises a cover sheet releasably adhered to atleast a portion of the bottom member, the cover sheet beingsubstantially impermeable to bacteria and water vapor. The cover sheetmay be coated with a gelling agent and/or nutrient mixture in the formof, for example, the above-described cold-water-soluble powder adheredto the cover sheet by means of an adhesive layer or a coating such asthat which is coated on the substrate of the body member in thisembodiment. Alternatively, the cover sheet may also be coated with onlya pressure-sensitive adhesive or may be free of any type of coating.Suitable materials for the cover sheet include those discussed above inconnection with the illustrated embodiments.

The material employed in the coating of this embodiment iscold-water-reconstitutable. As used in the specification and claims,"cold-water-reconstitutable" designates material which forms a solution,sol or gel in water at room temperature. Suitable gelling agents forinclusion in the coating of this embodiment (if such are contained inthe coating) include the above-described gelling agents which formsolutions in water at room temperatures. In addition, it has been foundthat agar, after it has been dissolved in boiling water and deposited asa coating, is a material which is "cold-water-resonstitutible".

A preferred coating mixture for providing the coating of this embodimentis prepared by mixing the following ingredients:

15 grams agar

32.7 grams peptone

16.3 grams yeast extract

6.5 grams dextrose

2.0 grams "Guar M150" (a polysaccharide, commercially available fromCelanese Corporation)

0.1 gram sodium carbonate

0.2 gram "Triton X-100" (a wetting agent, commercially available fromRohm and Haas)

1000 grams water

The coating may optionally include dyes, antibiotics and cross-linkingagents, examples of such ingredients including those describedhereinabove.

The body member of this embodiment may optionally comprise a spacerelement applied to the substrate, examples of suitable spacer elementsincluding those discussed above in connection with the illustratedembodiments. In the event such a spacer element is present, the coversheet may be, for example, releasably adhered to the spacer element.

The use of the devices of the present invention will be discussed withspecific reference to the device of FIGS. 1 and 3. To use the device ofFIGS. 1 and 3 as a pour plate, cover sheet 22 is pulled back and apredetermined quantity of water or an aqueous test sample is placed onsubstrate 12 of the body member. The gelling agent and/or nutrientsadhered to substrate 12 by adhesive 14 are quickly hydrated or dissolvedand a nutrient gel is formed. Cover sheet 22 is then replaced over thesubstrate, and a weighted plate placed on top to spread the samplecompletely. The device is then incubated for a predetermined period oftime. Any bacterial colonies growing in the medium can be countedthrough the transparent cover film.

The device may also be conveniently used for "Rodac" testing wherein thesurfaces of various objects are examined to determine the extent ofbacterial contamination. Cover sheet 22 coated only with apressure-sensitive adhesive is pulled back and touched to the surfacebeing tested. The adhesive picks up any microorganisms from the surfacebeing tested. The device is then hydrated, cover sheet 22 replaced, andthe device incubated.

The invention may be further illustrated by reference to the followingnon-limiting examples. All parts are expressed as parts by weight unlessotherwise indicated. The term "Standard Methods Nutrients" as usedherein refers to the nutrient mixture described in Standard Methods forthe Examination of Dairy Products, 14th Edition, American Public HealthAssociation, Washington, D.C. It consists of 5 parts peptone, 2.5 partsyeast extract and 1 part dextrose.

EXAMPLE 1

Transparent polyester film (0.018 inch thick, "Scotchpar" from 3M Co.)is coated with IOA/acrylamide (in a mole ratio of 94/6)pressure-sensitive adhesive at a level (measured when dry) of 2 grainsper 24 in² and dried. A "Volara" Type E polyethylene foam sheet(density: 6 lb/ft³, from Voltek Inc., Lawrence, Mass.) (0.06 inch thick)having side dimensions of 3 and 31/2 inches with a 2 inch diameter holecut out of the center is adhered to the dried adhesive side of the abovefilm. A mixture of 1 part Standard Methods Nutrients and 2 parts byweight guar gum powder HP-11 manufactured by Celanese Corp. is dusted onthe adhesive-coated film exposed by the cut out and the excess shakenloose. A cover sheet consisting of 0.0016 inch transparent biaxiallyoriented corona-treated polypropylene film is coated with the sameadhesive and coating weight used above, dried and dusted overall with amixture of one part triphenyl tetrazoleum chloride and 1500 parts byweight of xantham gum ("Keltrol" from Kelco Company, Chicago, Ill.). Theexcess powder is shaken loose. The sheet is cut to a dimension of 3×31/2inches and placed on the previously made laminate with the powder sidesfacing each other. The cover sheet and body member are heat-sealedtogether at one edge. The device, consisting of the three layers, issterilized in ethylene oxide. After suitable aeration the device isready for use and will remain so with reasonable care in storage formany months.

For use, the device is placed on a level surface and the top sheet islifted or removed. An aqueous test sample containing water (3 ml) iscarefully placed in the center of the cut-out and the cover sheetreplaced, powder side down. A slight weight may be applied to spread theliquid over the entire 2 inch cut-out. The device is placed in anincubator in the normal way. After incubation, the device is read forcolony growth just as a normal pour plate. The bacteria are dyed red foreasy quantitation.

EXAMPLE 2

A polyethylene-coated paper (0.0025 centimeter low density polyethyleneon the top and bottom sides of a bleached kraft paper, density: 90lb/320 yd² obtained from H. P. Smith) is printed with a 1 cm×1 cm blackgrid and a top varnish seal coat prior to the same adhesive coating ofExample 1. A polystyrene foam sheet identified as Valcour EPS, 0.038centimeter thick, 12 lbs/ft³ density, is cut to 3×31/2 inches with a 2inch diameter cut-out, and adhered as in Example 1 in place of thepolyethylene foam. The device is then powder coated, assembled andsterilized following the procedure of example 1. This device, when used,requires only a 1 ml water aliquot to hydrate and fill the 2 inchdiameter cut-out. After incubation the number of colonies can be read byreflected, rather than transmitted, light. The grid aids in the makingof an accurate count.

EXAMPLE 3-8

As indicated above, the preferred formulation of the dry medium of thisinvention uses guar gum, xantham gum and the Standard Methods Nutrients.Other cold-water-gelling agents may also be used. In the followingexamples, test samples of several gelling agents were made by mixing1500 grams of each agent and 1 gram of triphenyl tetrazoleum chloride.The mixtures were then coated onto a polypropylene tape (dimensions:3×31/2 inches, 1.6 mils thick) coated with a pressure-sensitive adhesiveof IOA/Acrylamide (in a mole ratio of 96/4) on one side. Mixing was doneby hand. Coating was done by shaking an excess of powder on the tape andbeating the excess off with a square rotating beater bar. These coatedtapes were used as cover sheets. The bottom sheets were coated withadhesive, guar gum and nutrients as described in Example 2.

The devices were inoculated with bacterial isolates from raw milk. Afterincubation at 35° C., the plates were examined by standard techniquesand the colonies counted. The results are tabulated in Table 1.

                  TABLE 1                                                         ______________________________________                                                                   24 hr.  48 hr.                                     Example No. Composition    Counts  Counts                                     ______________________________________                                        3           Agar           325     750                                                    Guar HP-11     250     700                                                    (Celanese)                                                        4           Guar CMHP      250     320                                                    (Celanese)                                                        5           CMC 7H         325     410                                                    (Hercules)                                                        6           Xanthan Gum    450     700                                                    (Kelco)                                                           7           Kelco HV Alginate                                                                            312     400                                                    (Kelco)                                                           8           Methocel 65HG  280     370                                                    (Dow)                                                             Control     Pour Plate     325     750                                        ______________________________________                                    

EXAMPLE 7

The established method of culturing used prior to the present inventionuses agar gel in petri dishes. The following experiment compares thisinvention with the petri dish pour plate technique known as the StandardMethods procedure (Standard Methods for the Examination of DairyProducts, 14th Edition, American Public Health Association, Washington,D.C., pages 87-03).

In this experiment, 24 samples of raw milk (Dairy Quality ControlInstitute, 2353 Rice Street, St. Paul, Minn.) were tested using thedevice of this invention and the Standard Methods procedure. The deviceof the present invention contained guar gum/Standard Methods Nutrientsin a 15/8 ratio by weight on the substrate and xantham gum/triphenyltetrazolium chloride in a 1500/1 ratio on the cover sheet. The StandardMethods procedure utilizes Standard Methods Agar (BBL Co.).

The correlation coefficient between the results obtained using thepresent invention and the results obtained by the prior art procedurewas 0.97. This test shows that the present invention provides increasedefficiency and convenience without sacrificing accuracy.

EXAMPLE 10

This experiment was done in a similar manner as in 9 above, except thatdifferent bacteria suspensions were used in place of standard milksamples. The bacteria inoculant suspensions had a concentration ofapproximately 1×10² CFU/ml (colony forming units per milliliter). Thestandard pour plate test was run according to the Standard Methodsprocedure (Standard Methods for the Examination of Dairy Products,American Public Health Association, pages 87-93). Results are in TableII.

                  TABLE II                                                        ______________________________________                                                    This Invention                                                                            CFU/ml Standard                                       Bacteria    CFU/ml      Methods Procedure                                     ______________________________________                                        Salmonella  220         300                                                   E. Coli     35          17                                                    Klebsiella  55           9                                                    S. aureus   80          85                                                    Pseudomonas 120         350                                                   S. epidermidis                                                                            155         220                                                   B. subtilis  1           1                                                    S. marcescens                                                                             340         250                                                   Shigella    13          15                                                    S. pyogenes 15          16                                                    E. cloacae  32          35                                                    ______________________________________                                    

EXAMPLE 11

Transparent, corona treated, biaxially-oriented polypropylene film(0.0016 inch thick) was coated with IOA/acrylamide (in a mole ratio of94/6) pressure-sensitive adhesive at a level (measured when dry) of 2grains per 24 in² and the adhesive layer was dried. Thepressure-sensitive adhesive also contained 0.0006 grams of2,3,5-triphenyl tetrazolium chloride per gram of dry adhesive. "GuarMeyprogat 150" (a polysaccharide commercially available from Celanese)was dusted on the adhesive-coated film at a level of 6 grains per 24in². Onto the layer of powder was coated a 20% solids solution ofStandard Methods Nutrients broth, the broth being dried to provide acoating weight of 2 grains per 24 in².

Devices were prepared using sheets of this material as both the bottommember and the cover sheet. The devices were inoculated with 1 cc. ofappropriate dilutions of bacteria and incubated 48 hours at 32° C.Results were compared to the results observed using the standard methodsprocedure, the results appearing in Table III below:

                  TABLE III                                                       ______________________________________                                                    The Device of                                                                             CFU/ml                                                            this Example                                                                              Standard Methods                                      Bacteria    CFU/ml      Procedure                                             ______________________________________                                        E. coli     153         120                                                   S. aureus    71         131                                                   S. fecalis  129         107                                                   ______________________________________                                    

EXAMPLE 12

"Schoeller Type MIL" photoprint paper (commercially available fromSchoeller Pulaski) was coated at a level of 3 grains per 24 in²(measured when dry) with the following solution and dried:

    ______________________________________                                        Coating Solution                                                              Ingredient         Grams                                                      ______________________________________                                        Agar               15                                                         Peptone            32.7                                                       Yeast Extract      16.3                                                       Dextrose           6.5                                                        "Guar Meyprogat 150"                                                                             2.0                                                        Water              1000                                                       ______________________________________                                    

The coated photoprint paper forms the bottom member of the device.

The cover sheet is a powder-coated polypropylene film of the typedescribed in Example 11 above except that here the pressure-sensitiveadhesive comprises 0.0012 grams of 2,3,5-triphenyl tetrazolium chlorideper gram of dry adhesive.

The devices were inoculated with 1 cc. of appropriate dilutions of thebacterial cultures indicated in Table IV below. Results after 48 hoursincubation at 32° C. were as indicated in Table IV below. Results arealso included for the Standard Methods Procedure.

                  TABLE IV                                                        ______________________________________                                                    The Device of                                                                             CFU/ml                                                            this Invention                                                                            Standard Methods                                      Bacteria    CFU/ml      Procedure                                             ______________________________________                                        S. aureus   330         335                                                   P. fragi    128         275                                                   S. fecalis  111         116                                                   S. agalactiae                                                                             180         170                                                   S. cremoris .sup. 900.sup.a                                                                           .sup. 900.sup.a                                       E. coli     143         177                                                   B. subtilis  11          20                                                   Pseudomonas 120         130                                                   ______________________________________                                         .sup.a number of colonies estimated due to large number thereof.         

When the devices of this Example were inoculated with Examples of rawmilk, a 0.934 correlation coefficient was observed relative to standardagar plates which were similarly inoculated.

EXAMPLE 13

"Schoeller Type MIL" photoprint paper was coated at a level of 3 grainsper 24 in² (measured when dry) with the following solution and dried:

    ______________________________________                                        Coating Solution                                                              Ingredient           Grams                                                    ______________________________________                                        Peptone              90                                                       Yeast Extract        45                                                       Dextrose             18                                                       "Guar M150"          8                                                        Sodium Carbonate     0.7                                                      "Triton X-100" (a wetting agent,                                                                   0.2                                                      commercially available from                                                   Rohm and Haas Corp.)                                                          Water                1000                                                     ______________________________________                                    

The coated photoprint paper forms the bottom member of the device.

The cover sheet was the same as that employed as the cover sheet inExample 12.

The devices were inoculated with 1 cc. of appropriate dilutions of thebacterial cultures indicated in Table V below. Results after 48 hoursincubation at 32° C. were as indicated in Table V below. Results arealso included for the Standard Methods Procedure.

                  TABLE V                                                         ______________________________________                                                    The Device of                                                                             CFU/ml                                                            this Invention                                                                            Standard Methods                                      Bacteria    CFU/ml      Procedure                                             ______________________________________                                        S. aureus   30          30                                                    P. fragi    57          95                                                    S. fecalis  100         95                                                    S. agalactiae                                                                             83          67                                                    S. cremoris 104         101                                                   E. coli      8           6                                                    B. subtilis 165         195                                                   ______________________________________                                    

EXAMPLE 14

A device in accordance with the present invention was constructed whichconsisted of the coated photoprint paper of Example 12 as the bottommember. The cover sheet consisted of a transparent, corona treated,biaxially-oriented polypropylene film (0.0016 inch thick) which had beencoated with IOA/acrylamide (in a mole ratio of 94/6) pressure-sensitiveadhesive at a level (measured when dry) of 2 grains per 24 in². Thepressure-sensitive adhesive also contained 0.0012 grams of2,3,5-triphenyl tetrazolium chloride per gram of dry adhesive.

After inoculating the device of this Example with a 0.1 cc of a dilutionof E. Coli and incubating 24 hours, individual colonies were observed.

What is claimed is:
 1. A device for growing microorganisms, comprising:abody member comprising a self-supporting, waterproof substrate havingupper and lower surfaces; a layer of adhesive-coated on said uppersurface of said substrate, said adhesive being non-inhibitory to thegrowth of microorganisms; and a cold-water-soluble powder adhereduniformly to said adhesive, said powder comprising at least oneingredient selected from the group consisting of a gelling agent, one ormore nutrients for growing microorganisms, and a mixture thereof;saiddevice being free of matrixes which adversely affect visualization andisolation of bacterial colonies.
 2. The device according to claim 1,further comprising a cover sheet releasably adhered to at least aportion of said body member.
 3. The device according to claim 2, whereinsaid cover sheet comprises a transparent film.
 4. The device accordingto claim 3, wherein said film is selected from the group consisting ofpolyester, polyethylene, polypropylene, polystyrene and silicone.
 5. Thedevice according to claim 1, wherein said powder comprises a gellingagent in sufficient amount to provide a gel having a Brookfieldviscosity of at least 1500 cps when hydrated with a predetermined amountof water.
 6. The device according to claim 1, further comprising ahydrophobic spacer element adhered to said upper surface of saidsubstrate forming side walls to retain a predetermined amount of liquidin contact with said substrate.
 7. The device according to claim 6,wherein said spacer element comprises a hydrophobic foam sheet having ahole therein.
 8. The device according to claim 7, wherein said foam ispolystyrene or polyethylene.
 9. The device according to claim 1, whereinsaid substrate is a film selected from the group consisting ofpolyester, polypropylene, polyethylene and polystyrene.
 10. The deviceaccording to claim 9, wherein said film is about 0.001 to 0.015 inchthick.
 11. The device according to claim 1, wherein said substrate has agrid pattern printed thereon.
 12. The device according to claim 1,wherein said powder comprises one or more nutrients for growingmicroorganisms.
 13. The device according to claim 1, wherein said powdercomprises a gelling agent and one or more nutrients for growingmicroorganisms.
 14. The device according to claim 1, wherein saidgelling agent is selected from the group consisting of xanthum gum, guargum, carboxymethyl cellulose, hydroxyethyl cellulose, and algin.
 15. Thedevice according to claim 14, wherein said gelling agent is guar gum,xanthum gum or mixtures thereof.
 16. The device according to claim 1,wherein said adhesive is a pressure-sensitive adhesive.
 17. The deviceaccording to claim 16, wherein said adhesive is substantiallytransparent when wetted with water.
 18. The device according to claim17, wherein said adhesive is a copolymer of isooctyl acrylate andacrylamide in a mole ratio of 94:6, respectively.
 19. The deviceaccording to claim 1, wherein one of said powder and said adhesivecontains a dye which is metabolizable by microorganisms and which causessaid microorganisms to be colored or fluorescent.
 20. The deviceaccording to claim 19, wherein said dye is selected from the groupconsisting of triphenyltetrazolium chloride, p-tolyltetrazolium red,tetrazolium violet and veratryltetrazoleum blue.
 21. A device forgrowing microorganisms, comprising: a bottom member comprising aself-supporting water-proof substrate having upper and lower surfaceswith a coating adhered to at least a portion of said upper surface, saidcoating being substantially water-free and consisting essentially of acold-water-reconstitutable material comprising at least one ingredientselected from the group consisting of a gelling agent, one or morenutrients for growing microorganisms, and a mixture thereof; and a coversheet releasably adhered to at least a portion of said bottom member andcapable of substantially overlying said coating on said substrate, saidcover sheet being substantially impermeable to bacteria and water vaporand having a coating adhered to at least a portion of the surface ofsaid cover sheet facing said body member, said coating beingsubstantially water-free and consisting essentially of awater-reconstitutable material comprising at least one ingredientselected from the group consisting of a gelling agent, one or morenutrients for growing microorganisms, and a mixture thereof; said devicebeing free of matrixes which adversely affect visualization andisolation of bacterial colonies.
 22. The device according to claim 21,wherein said coating on said cover sheet consists essentially of acold-water-soluble powder adhered to said cover sheet by means of anadhesive, said adhesive being coated on said substrate and beingnon-inhibitory to the growth of microorganisms, and saidcold-water-soluble powder being adhered uniformly to said adhesive. 23.The device according to claim 22, wherein said coating on said substrateconsists essentially of a cold-water-soluble powder adhered to saidsubstrate by means of an adhesive, said adhesive being coated on saidsubstrate and being non-inhibitory to the growth of microorganisms, andsaid cold-water-soluble powder being adhered uniformly to said adhesive.24. The device according to claim 23, wherein said cold-water-solublepowder adhered to said substrate comprises a gelling agent and one ormore nutrients for growing microorganisms, and said cold-water-solublepowder adhered to said cover sheet comprises a gelling agent.
 25. Thedevice according to claim 23, wherein said adhesive-coating on saidsubstrate and said adhesive-coated on said cover sheet are bothpressure-sensitive adhesives.
 26. The device according to claim 22,wherein said coating on said substrate comprises a gelling agent and oneor more nutrients for growing microorganisms, and saidcold-water-soluble powder adhered to said cover sheet comprises agelling agent.
 27. The device according to claim 22, further comprisinga hydrophobic spacer element adhered to said upper surface of saidsubstrate forming side walls to retain a predetermined amount of liquidin contact with said substrate.
 28. The device according to claim 22,wherein said adhesive is a pressure-sensitive adhesive.
 29. The deviceaccording to claim 21, further comprising a hydrophobic spacer elementadhered to said upper surface of said substrate forming side walls toretain a predetermined amount of liquid in contact with said substrate.30. The device according to claim 21, wherein said coating comprises agelling agent selected from the group consisting of xanthum gum, quargum, carboxymethyl cellulose, hydroxyethyl cellulose and algin.
 31. Thedevice according to claim 21, wherein said coating comprises a gellingagent selected from guar gum, xanthum gum and mixtures thereof.
 32. Thedevice according to claim 21, wherein said substrate is selected fromthe group consisting of a polyester film, polypropylene film,polyethylene film, polystyrene film, and polyethylene-coated paper. 33.The device according to claim 21, wherein said substrate is selectedfrom the group consisting of a film and a paper which has a water-proofcoating thereon, and said cover sheet is a film.